Point-of-sampling detection of Human Papillomavirus (HPV) Foundation for Applied Molecular Evolution Steven A. Benner Ozlem Yaren Human papillomavirus (HPV) infection is associated with cervical and oral cancers. Early detection with one of over 100 molecular tests, costing $100-200 each, is responsible for a substantial decrease in HPV-caused death in the developed world. Public health communities are eager to see this success mirrored in the developing world. More widespread screening will further slow the spread of this STD, and further reduce fatality from it, if the assays are less expensive, return results in minutes rather than weeks, and yield greater patient compliance among men and women. This project will deliver the next generation of HPV diagnostics to move the test down the food chain towards rapid (<60 min) calling of results at points-of-sampling, in doctors' offices, and in rural areas lacking doctors. The last is especially important in developing countries, where cost is a big constraint. This rules out polymerase chain reaction (PCR) to amplify HPV DNA in favor of isothermal amplifications. The goal of this R21 is to assess the ability of an innovative variant of the loop amplification (LAMP) isothermal amplification architecture, artificially expanded genetic information systems (AEGIS), and other innovations in nucleic acid chemistry, to deliver this product. Here, development of a point-of-sampling kit to detect HPV variants will go hand-in-hand with its use in a truly low resource environment, a reference laboratory run (at no cost to this grant) by Prof. Dr. Xian-Ping Ding at the Sichuan University in China. His laboratory will use fully protected patient samples to compare results from our kit with gold standard kits, and return results with complaints and suggestions. Development work here allows this project to proceed on an The Aims are to a large extent de-risked by R21 budget, presses us to have realistic costs, and gives us access to rural areas that are truly low resource, in a sense that exists nowhere in the US. work just published, where the Benner lab created a kit that detects dengue, chikungunya, and/or Zika in a single assay, after 30 minutes, in a single mosquito carcass, captured on Q-paper. The sample is rendered free of biohazard by treatment with ammonia and ethanol, and then dropped in a pre-prepared tube containing lyophilized reagents; these are shipped without refrigeration. Alternatively, urine or plasma is sampled (one drop). After being heated at 65 C, the assay results are viewed by reading through fluorescence output generated by blue LEDs mounted in a $10 device. The assay itself costs less than a dollar, has <1% false positives and <1% false negatives, and is presently being manufactured under a contract in Florida for environmental surveillance. The final deliverable will be a statement about the feasibility of the kit, useable by individuals who are trained in its use but not otherwise credentialed or licensed, to, within 30 minutes, tell if a mucosal swab contains one of two high risk HPV variants, and one or more low risk HPV strains.